Semen consists of a mixture of particulates including spermatozoa, other cells and a liquid portion, which comprises proteins, hormones and other dissolved or suspended molecules collectively known as seminal plasma. Seminal analysis and evaluation is a routine diagnostic procedure that is carried out in connection with fertility studies. Specimens should be analyzed within two hours of collection which places a time constraint on the analytical technique.
The analysis of semen involves the separation of the solid content from the liquid material. This has been done in the prior art by mechanical centrifugation using conventional techniques and an applied force of 500 to 1000 g for 10 to 30 minutes. The difficulty in separating the contents of semen using this type of a procedure is that the separated seminal plasma must be manually withdrawn from the centrifuge container before removal of the pellet which contains the separated spermatozoa. The pellet requires further washing to remove traces of the seminal plasma and resuspension in a suitable liquid. Analyses which require the separation of spermatozoa from seminal plasma include the determination of acrosin (Mohsenian et al., 1982 Fertil. Steril., 37:223-229; Goodpasture et al., J. Reprod. Fertil., 1981, 63:397-405; Goodpasture et al., J. Androl. 1982, 3:151-156; Goodpasture et al., J. Androl. 1987, 8:267-271; Schill. Fertil. Steril. 1975, 26:711-720; Schill et al., Int. J. Fertil. 1974, 19:217-227; Zaneveld et al., J. Reprod. Fertil. 1973, 32:525-529, all of which are incorporated by reference); hyaluronidase; acid phosphatases; .beta.-galactosidase; .beta.-N-acetylgalactosaminidase; .beta.-galactosidase (Mack et al. 1983, Biol. Reprod., 28:1032-1042) and anti-sperm antibodies (WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucous Interaction, 1987, Cambridge Univ. Press NY, N.Y., which is incorporated by reference). Seminal plasma is usually used for the determination of zinc, citric acid, fructose (all described in WHO Laboratory Manual), hormones such as relaxin (Weiss et al. 1986, Am. J. Obstetr. Gynecol. 154: 749-755) or metabolites such as glycerophosphocholine (Chap et al. 1988, Clin. Chem. 34: 106-109).
The centrifugation which is used in the separations that are required for seminal analyses is time consuming and difficult for running multiple samples. The required sample size of about 1.0 ml makes it impossible to analyze the same semen sample that is used for in vitro fertilization or artificial insemination.
Aside from the drawbacks of centrifugation, several assays for anti-sperm antibodies also have weaknesses. These assays use 1) agglutination (reviewed in R. Bronson et al. Fertil. Steril. (1984) 42: 171-183) which is very subjective and may give false negative and false positive results, 2) microscopic examination and counting of beads on the sperm surface (Bronson et al. 1982. Arch. Androl. 9: 61) which is labor-intensive and somewhat subjective, or 3) enzyme immunoassay of immobilized sonicated sperm including both intracellular and cell-surface antigens (W. Harel and D. Nelken. 1985. Am. J. Reprod. Immunol. Microbio. 8:137) which can give false-positive and false-negative results.